Hey this is my first try to analyse a rna-seq project. Since the company we worked with is not able to give me a usefull annotated differential expression table...
I just want to know for sure, if its half-way right what I do.
My samples: 2 conditions, 2 replicats from each condition, 50bp single-end, not strand-specific. I got 23m-50m reads per library.
I want to know differential expressed genes between conditions.
I first wanted to use bowtie2 for alignment and that worked pretty well until I noticed that no NH tag for htseqcount is written.
So I switched to tophat and there it got complicated:
In default tophat2 finds lesser alignments than bowtie2. Why? As I understand tophat2 uses bowtie2 for aligment.
As I dont want to find novel junctions, as there is no splicing my bacterium, the final command I used after several attempts is:
tophat2 -G file.gtf --no-novel-juncs --no-coverage-search --library-typ fr-unstranded index file.fastq
With every attempt (first: --no-coverage-search; second: added -G; third: added --no-novel juncs) the count of aligned reads dropped a little bit. Why it droppend between this 3 modes?
With the last mode I got an alignment rate of 65-75%.
I finally got my count tables with
samtools view file.bam | htseq-count -t gene -s no - file.gtf > counts.txt
Now I will use deseq for differential expression.
Everything ok so far?
I just want to know for sure, if its half-way right what I do.
My samples: 2 conditions, 2 replicats from each condition, 50bp single-end, not strand-specific. I got 23m-50m reads per library.
I want to know differential expressed genes between conditions.
I first wanted to use bowtie2 for alignment and that worked pretty well until I noticed that no NH tag for htseqcount is written.
So I switched to tophat and there it got complicated:
In default tophat2 finds lesser alignments than bowtie2. Why? As I understand tophat2 uses bowtie2 for aligment.
As I dont want to find novel junctions, as there is no splicing my bacterium, the final command I used after several attempts is:
tophat2 -G file.gtf --no-novel-juncs --no-coverage-search --library-typ fr-unstranded index file.fastq
With every attempt (first: --no-coverage-search; second: added -G; third: added --no-novel juncs) the count of aligned reads dropped a little bit. Why it droppend between this 3 modes?
With the last mode I got an alignment rate of 65-75%.
I finally got my count tables with
samtools view file.bam | htseq-count -t gene -s no - file.gtf > counts.txt
Now I will use deseq for differential expression.
Everything ok so far?
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